This invention relates to plant disease resistance, in particular to plant genes conferring pathogen resistance.
Plants are hosts to thousands of infectious diseases caused by a vast array of phytopathogenic fungi, bacteria, viruses, and nematodes. Plants recognize and resist many invading phytopathogens by inducing a rapid defense response, termed the hypersensitive response (HR). HR results in localized cell and tissue death at the site of infection, which constrains further spread of the infection. This local response often triggers non-specific resistance throughout the plant, a phenomenon known as systemic acquired resistance (SAR). Once triggered, SAR provides resistance for days to a wide range of pathogens. The generation of the HR and SAR in a plant depends upon the interaction between a dominant or semi-dominant resistance (R) gene product in the plant and a corresponding dominant avirulence (Avr) gene product expressed by the invading phytopathogen. It has been proposed that phylopathogen Avr products function as ligands, and that plant R products function as receptors. Thus, in the widely held model of phytopathogen/plant interaction, binding of the Avr product of an invading pathogen to a corresponding R product in the plant initiates the chain of events within the plant that produces HR and SAR and ultimately leads to disease resistance.
The production of transgenic plants carrying a heterologous gene sequence is now routinely practiced by plant molecular biologists. Methods for incorporating an isolated gene sequence into an expression cassette, producing plant transformation vectors, and transforming many types of plants are well known. Examples of the production of transgenic plants having modified characteristics as a result of the introduction of a heterologous transgene include: U.S. Pat. No. 5,719,046 to Guerineau (production of herbicide resistant plants by introduction of bacterial dihydropteroate synthase gene); U.S. Pat. No. 5,231,020 to Jorgensen (modification of flavenoids in plants); U.S. Pat. No. 5,583,021 to Dougherty (production of virus resistant plants); and U.S. Pat. No. 5,767,372 to De Greve and U.S. Pat. No. 5,500,365 to Fischoff (production of insect resistant plants by introducing Bacillus thuringiensis genes).
In conjunction with such techniques, the isolation of plant R genes has similarly permitted the production of plants having enhanced resistance to certain pathogens. Since the cloning of the first R gene, Pto from tomato, which confers resistance to Pseudomonas syringae pv. tomato (Martin et al., 1993), a number of other R genes have been reported (Hammond-Kosack and Jones, 1997). A number of these genes have been used to introduce the encoded resistance characteristic into plant lines that were previously susceptible to the corresponding pathogen. For example, U.S. Pat. No. 5,571,706 describes the introduction of the N gene into tobacco lines that are susceptible to Tobacco Mosaic Virus (TMV) in order to produce TMV-resistant tobacco plants. WO 95/28423 describes the creation of transgenic plants carrying the Rps2 gene from Arabidopsis thaliana, as a means of creating resistance to bacterial pathogens including Pseudomonas syringae, and WO 98/02545 describes the introduction of the Prf gene into plants to obtain broad-spectrum pathogen resistance.
Bacterial spot disease of tomato and pepper, caused by the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv), can be devastating to commercial production of these crops in areas of the world with high humidity and heavy rainfall. While control of Xcv in commercial agriculture is based largely on the application of pesticides, genetic resistance to bacterial spot disease has been described in both tomato and pepper (Cook and Stall, 1963; Cook and Guevara, 1984; Kim and Hartmann, 1985; Jones and Scott, 1986). Of the two hosts, genetic resistance in pepper has been more well characterized. Several single loci (Bs1, Bs2, and Bs3) that confer resistance in a xe2x80x9cgene-for-genexe2x80x9d manner have been identified (Hibberd et al., 1987). Moreover, the corresponding avirulence genes (avrBs1, avrBs2, and avrBs3) have been cloned from Xcv (Swanson et al., 1988; Minsavage et al., 1990). Genetic and molecular characterization of these avirulence genes has provided a great deal of information concerning the interaction between Xcv and pepper (Kearney et al., 1988; Kearney and Staskawicz, 1990; Herbers et al., 1992; Van den Ackerveken et al., 1996).
Of particular interest is the interaction governed by the avirulence gene avrBs2 and the resistance gene Bs2. AvrBs2 was originally identified as a 2.3 kb DNA fragment located on the Xcv chromosome (Minsavage et al., 1990; Kearney, 1989). Recently, it was established that avrBs2 encodes a protein with some homology to A. tumefaciens agrocinopine synthase and E. coli UgpQ, suggesting a possible enzymatic function (Swords et al., 1996). Mutant Xcv strains in which the avrBs2 gene has been disrupted or replaced are less virulent on susceptible hosts and are only able to grow to levels similar to that of wild type strains in a resistant host (Kearney, 1989; Kearney and Staskawicz, 1990; Swords et al., 1996). In addition, a survey of various races of Xcv and other pathovars of X. campestris has shown that avrBs2 is very widespread (Kearney and Staskawicz, 1990). For example, avrBs2 activity was shown to be present in Xc campestris (the causative agent of black rot in crucifers), Xc oryzae (now termed X. oryzae pv. oryzae (the causative agent of bacterial blight in rice), Xc citri (now termed X. axonopodis (the causative agent of citrus canker) and Xc phaseoli (the causative agent of bacterial blight of bean) (Kearney and Staskawicz, 1990). These studies also suggest that avrBs2 plays a highly conserved role in the fitness of X. campestris; isolates having avrBs2 show enhanced vigor on susceptible plant lines. The effectiveness of the Bs2 resistance gene against the some of the major races of Xcv appears to be based on this dual phenotype (fitness and elicitation of Bs2-mediated HR response) of the avrBs2 gene (Kearney and Staskawicz, 1990).
The availability of the Bs2 gene would facilitate the production of transgenic plants having resistance to a potentially wide range of phylopathogens. It is to such a gene that the present invention is directed.
The invention provides an isolated Bs2 gene from pepper that is shown to confer resistance to Xanthomonas campestris pv. vesicatoria when introduced into plants that are otherwise susceptible to infection by this organism. Specifically, such plants develop a hypersensitive response to the pathogen at the site of inoculation and show an enhanced resistance to systemic infection.
In one aspect of this invention, the nucleic acid sequences of the Bs2 gene and cDNA from pepper are provided, as is the amino acid sequence of the pepper Bs2 protein. The functional hallmark of the Bs2 protein is that it has Bs2 biological activity: when co-expressed in a plant with a Xanthomonas campestris AvrBs2 gene product, it produces a localized hypersensitive response, as determined by a transient assay technique described in detail below.
The existence of Bs2 homologs in other plant species including tomato and tobacco is demonstrated, and the invention provides the structural features and functional characteristics of such homologs and teaches how they may be isolated. Because the pepper Bs2 gene is the first Bs2 gene to have been isolated, it is referred to as the prototypical Bs2 gene; and the pepper Bs2 protein is referred to as the prototypical Bs2 protein. Homologs of the Bs2 protein are proteins that possess Bs2 biological activity and share a specified level of sequence identity with the prototype pepper Bs2 protein (typically at least 50% amino acid sequence identity). Nucleic acid molecules that encode such homologs are also encompassed by the invention.
In another aspect of the invention, nucleic acid molecules are provided that comprise a minimum number of consecutive nucleotides of the disclosed Bs2 sequences (e.g., at least 15 consecutive nucleotides of the pepper Bs2 cDNA). These molecules are useful, among other things, as PCR primers for amplifying portions of a Bs2 nucleic acid molecule, as sequencing primers to verify the authenticity of an amplified molecule, and as hybridization probes.
The invention also provides recombinant nucleic acid molecules that comprise a promoter sequence operably linked to a Bs2 open reading frame. Such molecules may be introduced into plants so as to confer enhanced resistance to phytopathogens such as Xanthomonas campestris. Transgenic plants comprising these molecules are also provided by the invention. Plants that may be usefully transformed with a Bs2 nucleic acid molecules to confer enhanced pathogen resistance include pepper, tomato, tobacco, broccoli, cauliflower, cabbage, cowpea, canola, bean, soybean, rice, corn, wheat, barley, citrus, cotton, cassava, grape and walnut.